Apoptosis has been identified in various cellular compartments of developing teeth and in teeth damaged by bacterial infection or operative procedures. It is not clear yet how apoptosis regulates odontogenesis, especially dentinogenesis. Our long-term goal is to understand how manipulation of apoptosis in teeth impacts odontogenesis;our specific objective of this application is to determine how apoptosis affects primary and reparative dentinogenesis. The central hypothesis of our application is that preventing odontoblast apoptosis by Bcl-2 overexpression impairs primary dentinogenesis but promotes reparative dentinogenesis. The rationale for the proposed research is that, once it is known how apoptosis influences odontogenesis, the process can be either up- or down-regulated via innovative approaches to preserving damaged teeth or creating new teeth for treating various dental diseases. With respect to anticipated outcomes, the work will demonstrate the role of odontoblast apoptosis in dentin formation and tooth damage repair. Such results will have an important positive impact on dental research, because the identified mechanisms are expected to provide new targets for therapeutic interventions that will help a large population of people who suffer of the consequences of damaged or missing teeth. In addition, we anticipate that our results will advance the fields of dental developmental biology and regenerative medicine. To test our central hypothesis, the following three specific aims will be pursued: 1) to evaluate whether overexpression of Bcl-2 in odontoblasts prevents their apoptosis;2) to evaluate how overexpression of Bcl-2 affects maturation and mineralization of odontoblasts;3) to evaluate whether overexpression of Bcl-2 in odontoblasts promotes reparative dentinogenesis induced by artificial cavities. We will utilize an available transgenic mouse model, Col2.3Bcl-2 (human Bcl-2 is driven by the 2.3 kb fragment of rat type I collagen promoter), to conduct our investigations. Additionally, we will use artificial cavity preparations to induce odontoblast apoptosis and reparative dentin formation, and a deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and detection of the large fragment (19 kDa) of cleaved caspase 3 to evaluate odontoblast apoptosis. We will evaluate odontoblast proliferation, differentiation, and mineralization in coronal pulp-derived cell cultures. Micro-CT will be used to assess dentin structures in vivo. Reparative dentinogenesis will be evaluated histologically and radiographically. The research proposed in this application is significant because it is expected to provide the knowledge required to develop alternative strategies to maintain the vitality and function of damaged teeth and produce bioengineered teeth with a controlled morphology. It is also anticipated to broaden our fundamental understanding of dentinogenesis under physiological and pathological conditions, improve the clinical success rate of various restorative procedures, preserve traumatized or infected teeth which otherwise would have to be devitalized or extracted, and ultimately improve the dental health status of our rapidly aging community. Public Health Relevance: This proposal will demonstrate the role of odontoblast apoptosis in dentin formation and tooth damage repair. It is expected to generate new interventions to preserving the vitality and functions of teeth injured by infections or trauma, and contributing to fabricated teeth via bioengineering.